This invention relates generally to microfluidic devices, particularly to devices used for analysis of biological samples, such as blood, urine and the like. These microfluidic devices bring small amounts of a liquid sample into contact with reagents to provide a qualitative or quantitative measure of the presence or absence of an analyte of interest. Typically, a measured amount of the sample is moved through one or more chambers containing reagents or conditioning agents used to prepare the sample for contacting the reagents. The amount of the sample is usually less than 10 μL and the chambers are of a similar size. They are interconnected by capillary passageways through which the sample moves by capillary forces or by an applied force, such as centrifugal force.
In many cases, it is necessary to contact the sample with a conditioning liquid in order to dilute the sample or otherwise prepare the sample for subsequent reaction. For example, assays often require a sample be contacted to minimize interference, to control reaction conditions such as pH, co-factors or ionic strength, to form complexes such as multi-dentate ligands, proteins such as antibody-antigen complexes, nucleic acids, polycarbohydrates, lipids or metals, to lysis cells e.g. bacteria, red blood cells or white blood cells, and to react analytes and metabolites into detectable form. Mixing of the sample with a conditioning liquid presents problems related to the small size of the microfluidic device. Movement of small amounts of liquids through narrow passageways by capillary forces involves the interaction of the liquid with the walls of the passageways. If the liquid is aqueous, which is typical of biological samples, and the walls of the passageway are hydrophilic and narrow, for example 200 to 200 μm wide and 1 to 200 μm deep, the surface energy of the liquid creates a force which can move the liquid through the passageway. The large surface to volume ratio means that the surface effects on the liquid are large. The Reynolds Number, a dimensionless unit which is related to the character of the liquid flow, is very low, indicating that the liquid flow is laminar, and not turbulent. Laminar flow is streamline flow, with the velocity increasing with the distance from the wall.
Mixing of a sample with conditioning liquids is difficult when laminar flow predominates. Mixing is usually done by creating turbulent conditions. In much of the prior art relating to microfluidics, liquids in laminar flow are brought into close contact, relying on diffusion of molecules from one layer of liquid to another to create a mixture of the liquids. In active micro mixers that use macro scale techniques e.g., mechanical stirring, including active elements can require very complex and costly devices.
In U.S. Pat. No. 6,136,272, Weigl et al disclose a device that creates two or more shallow laminar layers to facilitate the diffusion of molecules from one layer to an adjacent layer. The patentees stated that their device was designed so that the Reynolds Number is below 1, preferably less than 0.1. They observed that when the Reynolds Number is greater than 1, flow can be laminar, but that such systems are prone to developing turbulence when the flow pattern is disturbed. Thus, the patentees system was designed to assure laminar flow with diffusional mixing. Enhanced diffusion is created between parallel streams in laminar flow in another U.S. Published Patent Application 2002/0076300 (Weigl et al.).
U.S. Published Patent Application 2002/0097532 disclosed a disc containing many channels. Two liquids were passed through a zig-zag channel in laminar flow while the disc was rotated, with mixing said to occur by diffusion.
A T-Sensor is shown in U.S. Published Patent Application 2001/0042712. The sensor contacts a liquid sample with an indicator liquid, the streams flowing in parallel laminar flow with diffusion between them.
U.S. Published Application 2001/0048637 discloses a similar device, which overcomes the “butterfly effect” caused by greater diffusion at the walls than in the center of the parallel laminar flow streams.
U.S. Published Application 2002/0076350 illustrates another method of improving diffusion between laminar flow streams. Parallel laminar flow streams were moved through 90° turns to change the aspect ratio of the streams, thereby improving diffusion between the streams.
Micro-mixers are described in U.S. Pat. No. 6,190,034 B1 and U.S. Pat. No. 6,241,379 B1. Liquids are mixed by creating thin layers to facilitate mixing by diffusion.
The patents and applications discussed above are related to passing a reagent stream adjacent to a sample stream so that by diffusion a reaction occurs and then is measured. In other patents and applications mixing is attempted by various means, despite the liquids being in laminar flow.
In U.S. Published Application 2001/0048900 mixing separate streams by creating a vortex in a chamber. In some embodiments, the inventors indicate that a Reynolds number of 320 is achieved and the first and second fluids have Reynolds numbers between 1 and 2000. Therefore, the flow is in a region between laminar flow and turbulent flow.
U.S. Pat. No. 5,921,678 discloses a liquid mixer in which two streams of liquid meet head-on and exit together in a channel at 90° from the entrance channels. The Reynolds number of the streams is said to be 2000-6000. Sharp-edged pillars are shown to assist in generating turbulence at the intersection of the mixing streams.
U.S. Published Application 2002/0048535 shows a device in which two liquids are combined during rotation of the device to transfer the liquids from one container to another.
U.S. Pat. No. 6,264,900 provides mixing of parallel laminar flow streams for carrying out fast chemical reactions.
U.S. Pat. No. 6,065,864 discloses a micro-mixing system including bubble-controlled pumps and valves to establish circulating flow in a mixing chamber.
The present inventors wished to provide effective mixing of liquid reagents or conditioning liquids with sample fluids in microfluidic devices. Such mixing is made difficult by the mismatch between the viscosity and volume of the liquids to be mixed. Their solution to the problem will be described in detail below.